Macromolecular proton fraction (MPF) is a key biophysical parameter determining cross-relaxation between water and macromolecules in tissues. Over recent years, MPF has attracted significant interest as a potential biomarker of myelin in neural tissues. However, possible clinical applications of MPF have been hampered due to the absence of methods allowing fast and reliable in vivo measurements of this parameter. This project builds on our previous NIH-funded R21 study, where we developed, histologically validated, and clinically tested a principally new fast and robust method for whole-brain MPF mapping. This method achieves critical improvement in time efficiency by utilizing only one off-resonance saturation data point to measure MPF. The overall goal of this project is to develop a fast and clinically useful method for MPF mapping of the human spinal cord. The technical concept of the proposed method is based on the principle of single-point MPF mapping initially developed by our group for the brain imaging. However, implementation of this approach for the spinal cord imaging is challenging due to the small size of the anatomical structure of interest and motion problems. The project contains two specific aims. In the first aim, we will implement a series of technical solutions, which will allow improvements in spatial resolution and signal-to-noise ratio with simultaneous reduction of the scan time and motion sensitivity. Specifically, we will build the new spinal cord MPF mapping method on a combination of multi-echo summation, parallel imaging, reduced field-of-view acquisition, and a novel principle of synthetic reference, which is based on the replacement of an acquired reference image for data normalization by a calculated one derived from complimentary T1 and proton density maps. Based on these solutions, we will implement an MPF mapping protocol for the cervical and upper thoracic spinal cord with sub-millimeter spatial resolution and clinically affordable scan time. In the second aim, we will conduct a pilot study aimed to establish clinical utility of spinal cord MPF mapping in multiple sclerosis (MS). For this purpose, we will acquire MPF maps of the brain and spinal cord from a population of MS patients and healthy controls. We will further compare MPF in the spinal cord between MS patients and controls and between patients with relapsing-remitting and secondary-progressive disease courses, determine associations between spinal cord MPF and commonly accepted MS clinical status scales, and test the hypothesis that combined models including brain and spinal cord MPF better explain clinical status of MS patients than the models based on brain MPF alone. The MPF mapping method developed in this project is expected to be highly beneficial for disease diagnosis and treatment monitoring in conditions causing myelin damage in the spinal cord, such as MS, spinal cord injury, and cervical spondylotic myelopathy.